Process for L-amino acid production using Enterobacteriaceae by enhancing ahpC or ahpF encoding alkyl hydroperoxide reductase

ABSTRACT

A process for the preparation of L-amino acids, in particular L-threonine is disclosed herein. In particular, a process for the preparation of L-amino acids in Enterobacteriaceae, comprising fermenting, in a medium, Enterobacteriaceae which produce L-amino acid and in which  E. coli  ahpC and ahpF is overexpressed, and concentrating the L-amino acids in the medium or in the Enterobacteriaceae is disclosed.

This application is a 371 of PCT/EP02/06560 filed on Jun. 14, 2002 whichclaims benefit of 60/303,790 filed on Jul. 10, 2001.

FIELD OF THE INVENTION

This invention relates to a process for the preparation of L-aminoacids, in particular L-threonine, using strains of theEnterobacteriaceae family in which at least one or more of the geneschosen from the group consisting of ahpc and ahpF is (are) enhanced.

PRIOR ART

L-Amino acids, in particular L-threonine, are used in human medicine andin the. pharmaceuticals industry, in the foodstuffs industry and veryparticularly in animal nutrition.

It is known to prepare L-amino acids by fermentation of strains ofEnterobacteriaceae, in particular Escherichia coli (E. coli) andSerratia marcescens. Because of their great importance, work isconstantly being undertaken to improve the preparation processes.Improvements to the process can relate to fermentation measures, such ase.g. stirring and supply of oxygen, or the composition of the nutrientmedia, such as e.g. the sugar concentration during the fermentation, orthe working up to the product form, by e.g. ion exchange chromatography,or the intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used toimprove the output properties of these microorganisms. Strains which areresistant to antimetabolites, such as e.g. the threonine analogueα-amino-β-hydroxyvaleric acid (AHV), or are auxotrophic for metabolitesof regulatory importance and produce L-amino acid, such as e.g.L-threonine, are obtained in this manner.

Methods of the recombinant DNA technique have also been employed forsome years for improving the strain of strains of the Enterobacteriaceaefamily which produce L-amino acids, by amplifying individual amino acidbiosynthesis genes and investigating the effect on the production.

OBJECT OF THE INVENTION

The object of the invention is to provide new measures for improvedfermentative preparation of L-amino acids, in particular L-threonine.

SUMMARY OF THE INVENTION

The invention provides a process for the preparation of L-amino acids,in particular L-threonine, using microorganisms of theEnterobacteriaceae family which in particular already produce L-aminoacids and in which at least one or more of the nucleotide sequence(s)which code(s) for the ahpc and ahpF genes is (are) enhanced.

DETAILED DESCRIPTION OF THE INVENTION

Where L-amino acids or amino acids are mentioned in the following, thismeans one or more amino acids, including their salts, chosen from thegroup consisting of L-asparagine, L-threonine, L-serine, L-glutamate,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan and L-arginine. L-Threonine is particularly preferred.

The term “enhancement” in this connection describes the increase in theintracellular-activity of one or more enzymes or proteins in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or a gene or allele which codes. for a corresponding enzyme orprotein with a high activity, and optionally combining these measures.

By enhancement measures, in particular over-expression, the activity orconcentration of the corresponding protein is in general increased by atleast 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to amaximum of 1000% or 2000%, based on that of the wild-type protein or theactivity or concentration of the protein in the starting microorganism.

The process is characterized in that the following steps are carriedout:

-   -   a) fermentation of microorganisms of the Enterobacteriaceae        family in which one or more of the genes chosen from the group        consisting of ahpc and ahpF is (are) enhanced,    -   b) concentration of the corresponding L-amino acid in the medium        or in the cells of the microorganisms of the Enterobacteriaceae        family, and    -   c) isolation of the desired L-amino acid, constituents of the        fermentation broth and/or the biomass in its entirety or        portions (>0 to 100%) thereof optionally remaining in the        product.

The microorganisms which the present invention provides can produceL-amino acids from glucose, sucrose, lactose, fructose, maltose,molasses, optionally starch, optionally cellulose or from glycerol andethanol. They are representatives of the Enterobacteriaceae familychosen from the genera Escherichia, Erwinia, Providencia and Serratia.The genera Escherichia and Serratia are preferred. Of the genusEscherichia the species Escherichia coli and of the genus Serratia thespecies Serratia marcescens are to be mentioned in particular.

Suitable strains, which produce L-threonine in particular, of the genusEscherichia, in particular of the species Escherichia coli, are, forexample

-   -   Escherichia coli TF427    -   Escherichia coli H4578    -   Escherichia coli KY10935    -   Escherichia coli VNIIgenetika MG442    -   Escherichia coli VNIIgenetika M1    -   Escherichia coli VNIIgenetika 472T23    -   Escherichia coli BKIIM B-3996    -   Escherichia coli kat 13    -   Escherichia coli KCCM-10132.

Suitable L-threonine-producing strains of the genus Serratia, inparticular of the species Serratia marcescens, are, for example

-   -   Serratia marcescens HNr21    -   Serratia marcescens TLr156    -   Serratia marcescens T2000.

Strains from the Enterobacteriaceae family which produce L-threoninepreferably have, inter alia, one or more genetic or phenotypic featureschosen from the group consisting of: resistance toα-amino-β-hydroxyvaleric acid, resistance to thialysine, resistance toethionine, resistance to α-methylserine, resistance to diaminosuccinicacid, resistance to α-aminobutyric acid, resistance to borrelidin,resistance to rifampicin, resistance to valine analogues, such as, forexample, valine hydroxamate, resistance to purine analogues, such as,for example, 6-dimethylaminopurine, a need for L-methionine, optionallya partial and compensatable need for L-isoleucine, a need formeso-diaminopimelic acid, auxotrophy in respect of threonine-containingdipeptides, resistance to L-threonine, resistance to L-homoserine,resistance to L-lysine, resistance to L-methionine resistance toL-glutamic acid, resistance to L-aspartate, resistance to L-leucine,resistance to L-phenylalanine, resistance to L-serine, resistance toL-cysteine, resistance to L-valine, sensitivity to fluoropyruvate,defective threonine dehydrogenase, optionally an ability for sucroseutilization, enhancement of the threonine operon, enhancement ofhomoserine dehydrogenase I-aspartate kinase I, preferably of the feedback resistant form, enhancement of homoserine kinase, enhancement ofthreonine synthase, enhancement of aspartate kinase, optionally of thefeed back resistant form, enhancement of aspartate semialdehydedehydrogenase, enhancement of phosphoenol pyruvate carboxylase,optionally of the feed back resistant form, enhancement of phosphoenolpyruvate synthase, enhancement of transhydrogenase, enhancement of theRhtB gene product, enhancement of the RhtC gene product, enhancement ofthe YfiK gene product, enhancement of a pyruvate carboxylase, andattenuation of acetic acid formation.

It has been found that microorganisms of the Enterobacteriaceae familyproduce L-amino acids, in particular L-threonine, in an improved mannerafter enhancement, in particular over-expression, of at least one ormore of the genes chosen from the group consisting of ahpC and ahpF.

The use of endogenous genes is in general preferred. “Endogenous genes”or “endogenous nucleotide sequences” are understood as meaning the genesor nucleotide sequences present in the population of a species.

The nucleotide sequences of the genes of Escherichia coli belong to theprior art and can also be found in the genome sequence of Escherichiacoli published by Blattner et al. (Science 277: 1453–1462 (1997)).

The following information on the ahpc gene and ahpF gene is known, interalia, from the prior art:

-   ahpc gene:-   Description: C22 subunit of alkyl hydroperoxide reductase;    detoxification of hydroperoxides-   EC No.: 1.6.4.-   Reference: Ferrante et al.; Proceedings of the National Academy of    Sciences USA 92 (17): 7617–7621 (1995) Poole and Ellis; Biochemistry    35(1): 56–64 (1996) Nishiyama et al.; Journal of Bacteriology    183(8): 2431–2438 (2001)-   Accession No.: AE000166-   ahpF gene:-   Description: F52a subunit of alkyl hydroperoxide reductase;    detoxification of hydroperoxides-   Reference: Ferrante et al.; Proceedings of the National Academy of    Sciences USA 92(17): 7617–7621 (1995) Poole and Ellis; Biochemistry    35(1): 56–64 (1996) Poole et al.; European Journal of Biochemistry    267(20): 6126–6133 (2000)-   Accession No.: AE000166

The nucleic acid sequences can be found in the databanks of the NationalCenter for Biotechnology Information (NCBI) of the National Library ofMedicine (Bethesda, Md., USA), the nucleotide sequence databank of theEuropean Molecular Biologies Laboratories (EMBL, Heidelberg, Germany orCambridge, UK) or the DNA databank of Japan (DDBJ, Mishima, Japan).

Alleles of at least one or more of the genes chosen from the groupconsisting of ahpC and ahpF which result from the degeneracy of thegenetic code or due to “sense mutations” of neutral function canfurthermore be used.

To achieve an enhancement, for example, expression of the genes or thecatalytic properties of the proteins can be increased. The two measurescan optionally be combined.

To achieve an over-expression, the number of copies of the correspondinggenes can be increased, or the promoter and regulation region or theribosome binding site upstream of the structural gene can be mutated.Expression cassettes which are incorporated upstream of the structuralgene act in the same way. By inducible promoters, it is additionallypossible to increase the expression in the course of fermentativeL-threonine production. The expression is likewise improved by measuresto prolong the life of the m-RNA. Furthermore, the enzyme activity isalso enhanced by preventing the degradation of the enzyme protein. Thegenes or gene constructs can either be present in plasmids with avarying number of copies, or can be integrated and amplified in thechromosome. Alternatively, an over-expression of the genes in questioncan furthermore be achieved by changing the composition of the media andthe culture procedure.

Instructions in this context can be found by the expert, inter alia, inChang and Cohen (Journal of Bacteriology 134: 1141–1156 (1978)), inHartley and Gregori (Gene 13: 347–353 (1981)), in Amann and Brosius(Gene 40: 183–190 (1985).), in de Broer et al. (Proceedings of theNational Academy of Sciences of the United States of America 80: 21–25(1983)), in LaVallie et al. (BIO/TECHNOLOGY 11: 187–193 (1993)), inPCT/US97/13359, in Llosa et al. (Plasmid 26: 222–224 (1991)), in Quandtand Klipp (Gene 80: 161–169 (1989)), in Hamilton (Journal ofBacteriology 171: 4617–4622 (1989)), in Jensen and Hammer (Biotechnologyand Bioengineering 58: 191–195 (1998)) and in known textbooks ofgenetics and molecular biology.

Plasmid vectors which are capable of replication in Enterobacteriaceae,such as e.g. cloning vectors derived from pACYC184 (Bartolomé et al.;Gene 102: 75–78 (1991)), pTrc99A (Amann et al.; Gene 69: 301–315 (1988))or pSC101 derivatives (Vocke and Bastia, Proceedings of the NationalAcademy of Sciences USA 80(21): 6557–6561 (1983)) can be used. A straintransformed with a plasmid vector, wherein the plasmid vector carries atleast one or more of the genes chosen from the group consisting of ahpcand ahpF or nucleotide sequences which code for them, can be employed ina process according to the invention.

It is also possible to transfer mutations which affect the expression ofthe particular gene into various strains by sequence exchange (Hamiltonet al. (Journal of Bacteriology 171: 4617–4622 (1989)), conjugation ortransduction.

It may furthermore be advantageous for the production of L-amino acids,in particular L-threonine, with strains of the Enterobacteriaceae familyto enhance one or more enzymes of the known threonine biosynthesispathway or enzymes of anaplerotic metabolism or enzymes for theproduction of reduced nicotinamide adenine dinucleotide phosphate, inaddition to the enhancement of one or more of the genes chosen from thegroup consisting of ahpC and ahpF.

Thus, for example, one or more of the genes chosen from the groupconsisting of

-   -   the thrABC operon which codes for aspartate kinase, homoserine        dehydrogenase, homoserine kinase and threonine synthase (U.S.        Pat. No. 4,278,765),    -   the pyc gene which codes for pyruvate carboxylase (DE-A-19 831        609)    -   the pps gene which codes for phosphoenol pyruvate synthase        (Molecular and General Genetics 231(2): 332–336 (1992)),    -   the ppc gene which codes for phosphoenol pyruvate carboxylase        (Gene 31: 279–283 (1984)),    -   the pnta and pntB genes which code for transhydrogenase        (European Journal of Biochemistry 158: 647–653 (1986)),    -   the rhtB gene which imparts homoserine resistance (EP-A-0 994        190),    -   the mqo gene which codes for malate:quinone oxidoreductase (WO        02/06459),    -   the rhtC gene which imparts threonine resistance (EP-A-1 013        765),    -   the thrE gene of Corynebacterium glutamicum which codes for the        threonine export protein (WO 01/92545),    -   the gdhA gene which codes for glutamate dehydrogenase (Nucleic        Acids Research 11: 5257–5266 (1983); Gene 23: 199–209 (1983)),    -   the dps gene which codes for the global regulator Dps (Genes &        Development 6(12B): 2646–2654 (1992), Accession No. AE000183),    -   the hns gene which codes for the DNA-binding protein HLP-II        (Molecular and General Genetics 212(2): 199–202 (1988),        Accession No. AE000222),    -   the lrp gene which codes for the regulator of the leucine Lrp        regulon and high-affinity transport systems of branched-chain        amino acids (Journal of Biological Chemistry 266(17):        10768–10774 (1991), Accession No. AE000191),    -   the pgm gene which codes for phosphoglucomutase (Journal of        Bacteriology 176: 5847–5851 (1994), Accession No. AE000172),    -   the fba gene which codes for fructose bisphosphate aldolase        (Biochemical Journal 257: 529–534 (1989), Accession No.        AE000376),    -   the ptsG gene which codes for the glucose-specific IIBC        component of the phosphotransferase system PTS (Journal of        Biological Chemistry 261(35): 16398–16403 (1986), Accession No.        AE000210),    -   the ptsH gene of the ptsHIcrr operon which codes for the        phosphohistidine protein hexose phosphotransferase of the        phosphotransferase system PTS (Journal of Biological Chemistry        262(33): 16241–16253 (1987), Accession No. AE000329),    -   the ptsI gene of the ptsHIcrr operon which codes for enzyme I of        the phosphotransferase system PTS (Journal of Biological        Chemistry 262(33): 16241–16253 (1987), Accession No. AE000329),    -   the crr gene of the ptsHIcrr operon which codes for the        glucose-specific IIA component of the phosphotransferase system        PTS (Journal of Biological Chemistry 262(33): 16241–16253        (1987), Accession No. AE000329),    -   the mopB gene which codes for chaperone GroES (Journal of        Biological Chemistry 261(26): 12414–12419 (1986), Accession No.        AE000487), can be enhanced, in particular over-expressed.

The use of endogenous genes is in general preferred.

It may furthermore be advantageous for the production of L-amino acids,in particular L-threonine, in addition to the enhancement of one or moreof the genes chosen from the group consisting of ahpC and ahpF, for oneor more of the genes chosen from the group consisting of

-   -   the tdh gene which codes for threonine dehydrogenase (Journal of        Bacteriology 169: 4716–4721 (1987)),    -   the mdh gene which codes. for malate dehydrogenase (E.C.        1.1.1.37) (Archives in Microbiology 149: 36–42 (1987)),    -   the gene product of the open reading frame (orf) yjfA (Accession        Number AAC77180 of the National Center for Biotechnology        Information (NCBI, Bethesda, Md., USA)),    -   the gene product of the open reading frame (orf) ytfP (Accession        Number AAC77179 of the National Center for Biotechnology        Information (NCBI, Bethesda, Md., USA)),    -   the pckA gene which codes for the enzyme phosphoenol pyruvate        carboxykinase (Journal of Bacteriology 172: 7151–7156 (1990)),    -   the poxB gene which codes for pyruvate oxidase (Nucleic Acids.        Research 14(13): 5449–5460 (1986)),    -   the aceA gene which codes for the enzyme isocitrate lyase        (Journal of Bacteriology 170: 4528–4536 (1988)),    -   the dgsA gene which codes for the DgsA regulator of the        phosphotransferase system (Bioscience, Biotechnology and        Biochemistry 59: 256–251 (1995)) and is also known under the        name of the mlc gene,    -   the fruR gene which codes for the fructose repressor (Molecular        and General Genetics 226: 332–336 (1991)) and is also known        under the name of the cra gene, and    -   the rpoS gene which codes for the sigma³⁸ factor (WO 01/05939)        and is also known under the name of the katF gene,        to be attenuated, in particular eliminated or for the expression        thereof to be reduced.

The term “attenuation” in this connection describes the reduction orelimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or a gene or allele which codes fora corresponding enzyme with a low activity or inactivates thecorresponding enzyme (protein) or gene, and optionally combining thesemeasures.

By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to25%, 0 to 10% or 0 to 5% of the activity or concentration of thewild-type protein or of the activity or concentration of the protein inthe starting microorganism.

It may furthermore be advantageous for the production of L-amino acids,in particular L-threonine, in addition to the enhancement of one or moreof the genes chosen from the group consisting of ahpC and ahpF, toeliminate undesirable side reactions (Nakayama: “Breeding of Amino AcidProducing Microorganisms”, in: overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek.(eds.), Academic Press, London, UK, 1982).

The microorganisms produced according to the invention can be culturedin the batch process (batch culture), the fed batch (feed process) orthe repeated fed batch process (repetitive feed process). A summary ofknown culture methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einfhrüng in die Bioverfahrenstechnik [BioprocessTechnology 1. Introduction to Bioprocess Technology (Gustav FischerVerlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktorenund periphere Einrichtungen [Bioreactors and Peripheral Equipment](Vieweg Verlag, Braunschweig/wiesbaden, 1994)).

The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofmethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose,fructose, maltose, molasses, starch and optionally cellulose, oils andfats, such as e.g. soya oil, sunflower oil, groundnut oil and coconutfat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleicacid, alcohols, such as e.g. glycerol and ethanol, and organic acids,such as e.g. acetic acid, can be used as the source of carbon. Thesesubstances can be used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract,meat extract, malt extract, corn steep liquor, soya bean flour and urea,or inorganic compounds, such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, can be usedas the source of nitrogen. The sources of nitrogen can be usedindividually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used asthe source of phosphorus. The culture medium must furthermore comprisesalts of metals, such as e.g. magnesium sulfate or iron sulfate, whichare necessary for growth. Finally, essential growth substances, such asamino acids and vitamins, can be employed in addition to theabovementioned substances. Suitable precursors can moreover be added tothe culture medium. The starting substances mentioned can be added tothe culture in the form of a single batch, or can be fed in during theculture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acid compounds, such as phosphoric acid orsulfuric acid, can be employed in a suitable manner to control the pH ofthe culture. Antifoams, such as e.g. fatty acid polyglycol esters, canbe employed to control the development of foam. Suitable substanceshaving a selective action, e.g. antibiotics, can be added to the mediumto maintain. the stability of plasmids. To maintain aerobic conditions,oxygen or oxygen-containing gas mixtures, such as e.g. air, areintroduced into the culture. The temperature of the culture is usually25° C. to 45° C., and preferably 30° C. to 40° C. Culturing is continueduntil a maximum of L-amino acids or L-threonine has formed. This targetis usually reached within 10 hours to 160 hours.

The analysis of L-amino acids can be carried out by anion exchangechromatography with subsequent ninhydrin derivation, as described bySpackman et al. (Analytical Chemistry 30: 1190–1206 (1958), or it cantake place by reversed phase HPLC as described by Lindroth et al.(Analytical Chemistry 51: 1167–1174 (1979)).

The process according to the invention is used for the fermentativepreparation of L-amino acids, such as, for example, L-threonine,L-isoleucine, L-valine, L-methionine, L-homoserine and L-lysine, inparticular L-threonine.

The present invention is explained in more detail in the following withthe aid of embodiment examples.

The minimal (M9) and complete media (LB) for Escherichia coli used aredescribed by J. H. Miller (A Short Course in Bacterial Genetics (1992),Cold Spring Harbor Laboratory Press). The isolation of plasmid DNA fromEscherichia coli and all techniques of restriction, ligation, Klenow andalkaline phosphatase treatment are carried out by the method of Sambrooket al. (Molecular Cloning—A Laboratory anual (1989) Cold Spring HarborLaboratory Press). Unless described otherwise, the transformation ofEscherichia coli is carried out by the method of Chung et al.(Proceedings of the National Academy of Sciences of the United States ofAmerica (1989) 86: 2172–2175).

The incubation temperature for the preparation of strains andtransformants is 37° C.

EXAMPLE 1

Construction of the Expression Plasmid pTrc99AahpCF

The ahpC and ahpF genes from E. coli K12 are amplified using thepolymerase chain reaction (PCR) and synthetic oligonucleotides. Startingfrom the nucleotide sequences of the ahpC and ahpF genes in E. coli K12MG1655 (Accession Number AE000166, Blattner et al. (Science 277:1453–1462 (1997)), PCR primers are synthesized (MWG Biotech, Ebersberg,Germany). The sequences of the primers are modified such thatrecognition sites for restriction enzymes are formed. The recognitionsequence for XbaI is chosen for the ahpCF1 primer and the recognitionsequence for HindIII for the ahpCF2 primer, which are marked byunderlining in the nucleotide sequence shown below:

ahpCF1: 5′ - GCATCTAGACGATAACACGGAGGAAG - 3′ (SEQ ID No. 1) ahpCF2: 5′ -GCTAAGCTTTTGCAGGTGAATC - 3′ (SEQ ID No. 2)

The chromosomal E. coli K12 MG1655 DNA employed for the PCR is isolatedaccording to the manufacturer's instructions with “Qiagen Genomic-tips100/G” (QIAGEN, Hilden, Germany). A DNA fragment approx. 2400 bp in sizecan be amplified with the specific primers under standard PCR conditions(Innis et al. (1990) PCR-Protocols. A Guide to Methods and Applications,Academic Press) with Pfu-DNA polymerase (Promega Corporation, Madison,USA). The PCR product is cleaved with the restriction enzymes xbaI andHindIII and ligated with the vector pTrc99A (Pharmacia Biotech, Uppsala,Sweden), which has been digested with the enzymes XbaI and HindIII. TheE. coli strain XL1-Blue MRF (Stratagene, La Jolla, USA) is transformedwith the ligation batch and plasmid-carrying cells are selected on LBagar, to which 50 μg/ml ampicillin are added. Successful cloning can bedemonstrated after plasmid DNA isolation by control cleavage with theenzymes EcoRI and KuI. The plasmid is called pTrc99AahpCF (FIG. 1).

EXAMPLE 2

Preparation of L-threonine with the Strain MG442/pTrc99AahpCF

The L-threonine-producing E. coli strain MG442 is described in thepatent specification U.S. Pat. No. 4,278,765 and deposited as CMIMB-1628 at the Russian National Collection for Industrial Microorganisms(VKPM, Moscow, Russia).

The strain MG442 is. transformed with the expression plasmidpTrc99AahpCF described in. example 1 and with the vector pTrc99A andplasmid-carrying cells are selected on LB agar with 50 μg/ml ampicillin.The strains MG442/pTrc99AabpCF and MG442/pTrc99A are formed in thismanner. Selected individual colonies are then multiplied further onminimal medium with the following composition: 3.5 g/l Na₂HPO₄*2H₂O, 1.5g/l KH₂PO₄, 1 g/l NH₄Cl, 0.1 g/l MgSO₄*7H₂O, 2 g/l glucose, 20 g/l agar,50 mg/l ampicillin. The formation of L-threonine is checked in batchcultures of 10 ml contained in 100 ml conical flasks. For this, 10 ml ofpreculture medium of the following composition: 2 g/l yeast extract, 10g/l (NH₄)₂SO₄, 1 g/l KH₂PO₄, 0.5 g/l MgSO₄*7H₂O, 15 g/l CaCO₃, 20 g/lglucose, 50 mg/l-ampicillin are inoculated and the batch is incubatedfor 16 hours at 37° C. and 180 rpm on an ESR incubator from Kühner AG(Birsfelden, Switzerland).

250 μl portions of this preculture are transinoculated into 10 ml ofproduction medium (25 g/l (NH₄)₂SO₄, 2 g/l KH₂PO₄, 1 g/l MgSO₄*7H₂O,0.03 g/l FeSO₄*7H₂O, 0.018 g/l MnSO₄*1H₂O, 30 g/l CaCO₃, 20 g/l glucose,50 mg/l ampicillin) and the batch is incubated for 48 hours at 37° C.The formation of L-threonine by the starting strain MG442 isinvestigated in the same manner, but no addition of ampicillin to themedium takes place. After the incubation the optical density (OD) of theculture suspension is determined with an LP2W photometer from Dr. Lange(Düsseldorf, Germany) at a measurement wavelength of 660 nm.

The concentration of L-threonine formed is then determined in thesterile-filtered culture supernatant with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column reaction with ninhydrin detection.

The result of the experiment is shown in table 1.

TABLE 1 OD L-Threonine Strain (660 nm) g/l MG442 5.6 1.4 MG442/pTrc99A3.8 1.3 MG442/pTrc99AahpCF 5.4 2.6

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1: Map of the plasmid pTrc99AahpCF containing the ahpc and ahpFgenes.

The length data are to be understood as approx. data. The abbreviationsand designations used have the following meaning:

Amp: Ampicillin resistance gene lacI: Gene for the repressor protein ofthe trc promoter Ptrc: trc promoter region, IPTG-inducible ahpC: Codingregion of the ahpC gene ahpF: Coding region of the ahpF gene 5S: 5S rRNAregion rrnBT: rRNA terminator region

The abbreviations for the restriction enzymes have the following meaning

-   -   EcoRI: Restriction endonuclease from Escherichia coli RY13    -   HindIII: Restriction endonuclease from Haemophilus influenzae    -   MluI: Restriction endonuclease from Hicrococcus luteus IFO 12992    -   XbaI: Restriction endonuclease from Xanthomonas campestris

1. A process for preparing and quantifying L-amino acids in a medium orin an Enterobacteriaceae, comprising: a) fermenting, in a medium,Enterobacteriaceae which produce L-amino acid and in which E. coli ahpCand ahpF is overexpressed by increasing the copy number or by operablylinking to a promoter, and b) quantifying the L-amino acids in themedium or in the Enterobacteriaceae.
 2. A process according to claim 1,wherein at least one gene is additionally overexpressed, said at leastone gene is selected from the group consisting of: a) the thrABC operonwhich codes for encodes aspartate kinase, homoserine dehydrogenase,homoserine kinase and threonine synthase, b) the pyc gene which encodespyruvate carboxylase, c) the pps gene which encodes phosphoenol pyruvatesynthase, d) the ppc gene which encodes phosphoenol pyruvatecarboxylase, e) the pntA and pntB genes which encode transhydrogenase,f) the rhtB gene which encodes a protein which imparts homoserineresistance, g) the mqo gene which encodes malate:quinine oxidoreductase,h) the rhtC gene which encodes a protein which imparts threonineresistance, i) the thrE gene which encodes the threonine export protein,j) the gdhA gene which encodes glutamate dehydrogenase, k) the hns genewhich encodes the DNA-binding protein HLP-II, I) the lrp gene whichencodes the regulator of the leucine Lrp regulon, m) the pgm gene whichencodes phosphoglucomutase, n) the fba gene which encodes fructosebisphosphate aldolase, o) the ptsG gene which encodes theglucose-specific IIBC component, p) the ptsH gene which encodes thephosphohistidine protein hexose phosphotransferase, q) the ptsl genewhich encodes enzyme I of the phosphotransferase system, r) the crr genewhich encodes the glucose-specific IIA component, s) the mopb gene whichencodes chaperone GroES, and t) the dps gene which encodes the globalregulator Dps.
 3. A process according to claim 1, wherein at least onegene is eliminated or its expression is reduced, said at least one geneis selected from the group consisting of: a) the tdh gene which encodesthreonine dehydrogenase, b) the mdh gene which encodes malatedehydrogenase, c) the gene product of the open reading frame (orf) yjfA,d) the gene product of the open reading frame (orf) ytfP, e) the pckAgene which encodes phosphoenol pyruvate carboxykinase, f) the poxB genewhich encodes pyruvate oxidase, g) the aceA gene which encodesisocitrate lyase, h) the dgsA gene which encodes the DgsA regulator ofthe phosphotransferase system, i) the fruR gene which encodes thefructose repressor, and j) the rpoS gene which encodes the sigma38factor.
 4. A process according to claim 1, further comprising c)isolating the L-amino acids.
 5. A process according to claim 4, whereinat least a portion of constituents of the medium and/or a biomassresulting from fermenting remain with the isolated L-amino acids.
 6. Aprocess according to any one of claims 2, 3, 4, 5, and 1, wherein theL-amino acids are L-threonine, L-lysine or both.